Phosphate-Based Self-Immolative Linkers for the Delivery of Amine-Containing Drugs

Amine-containing drugs often show poor pharmacological properties, but these disadvantages can be overcome by using a prodrug approach involving self-immolative linkers. Accordingly, we designed l-lactate linkers as ideal candidates for amine delivery. Furthermore, we designed linkers bearing two different cargos (aniline and phenol) for preferential amine cargo release within 15 min. Since the linkers carrying secondary amine cargo showed high stability at physiological pH, we used our strategy to prepare phosphate-based prodrugs of the antibiotic Ciprofloxacin. Therefore, our study will facilitate the rational design of new and more effective drug delivery systems for amine-containing drugs.


Introduction
Drugs containing an amino group are key pharmaceutical agents, covering a broad spectrum of biological actions and displaying anti-inflammatory [1], anticancer [2,3], antimicrobial [4,5], and pain-relieving properties [6]. Currently, 542 drugs containing an amino group have already been approved for the EU market, according to the drug bank online (https://go.drugbank.com, accessed on 15 July 2021). This number does not include many other biologically active compounds-potential leads-or compounds from natural resources, such as alkaloids. However, amine drugs often show poor pharmacological properties, such as low aqueous solubility and poor membrane permeability due to ionization of the amino group [7] under physiological conditions. Nevertheless, amines are generally susceptible to derivatization. Thus, a prodrug strategy [8][9][10] can be used to overcome these drawbacks [7,11].
Prodrug strategies rely on a structural modification (masking) of the active pharmaceutical agent-a drug-with a suitable protecting group (promoiety) to modulate its pharmacokinetic properties. Such a change helps to facilitate drug delivery to the target site (e.g., tissues, cells, cell compartments, or organs). One of the most rapidly developing prodrug strategies consists of using self-immolative (SI) linkers [12,13] to control drug release [14].
SI linkers are covalent assemblies that couple an active compound (drug) to a protecting group. After external stimuli, either chemical or enzymatic, a cascade of spontaneous reactions [15] leads to linker fragmentation and consequently to drug release. The two main classes of SI linkers are (1) carbamates and (2) phosphate-based systems. Phosphorus-based SI linkers stand above the "classical" carbamate linkers because they make it possible to attach an additional substituent, which can help fine-tune the SI rate or provide a double cargo option.
Phosphorus-based SI linkers have been introduced as suitable drug-delivery systems for several drugs. A paradigmatic example of a phosphorus-based SI linker application is the methoxymethylphosphonic acid (MMPA) drug delivery vehicle for the oral delivery of propofol [16]. Other examples include pro-nucleotide prodrugs (ProTides) [17], which have been used to treat various viral infections, such as HIV [18], hepatitis B [19], or SARS-Cov-2 (COVID-19) [20]. Considering their success as drug-delivery systems, phosphatebased SI linkers may find broader applications in drug discovery and materials science through systematic studies.
Target linkers were synthesized in two consecutive phosphorylation steps, starting from commercially available phosphorodichloridates 20-21 (Scheme 1). Carbonate 19 was prepared in a reaction between ethylene glycol and dimethoxynitrobenzyl (DMNB) chloroformate in THF, using pyridine as the base [22]. The reaction of equimolar amounts of dichloridates 20-21 and DMNB carbonate 19 in the presence of triethylamine (TEA) in toluene gave intermediates 22 and 23. This reaction was monitored by 31 P-NMR spectra (signal at δ P 4.7 ppm for 22 and δ P 0.08 ppm for 23, in CDCl 3 ), and intermediates 22 and 23 were directly used for the second phosphorylation step. The reaction of 22 and 23 with one equivalent of the corresponding amine and TEA as a base afforded the final linkers 1-6. The isolated yields in the series bearing ethoxy substituent (1-3) were moderate (23-44%), whereas compounds 4-6 were isolated in low yields (4-12%) despite multiple flash chromatography (silica gel and C18). The self-immolation of 1-6 was triggered photochemically (365 nm), and the reaction was monitored by 31 P-NMR spectroscopy. Surprisingly, the successful photoactivation of 1-3 yielded intermediates (1-3)-I, with no further spectral change over several days, thus indicating that the cargo was not released ( Figure S1 in Supplementary Materials).
In contrast to 1-3, linkers 4 and 6 afforded cyclic intermediates 4-cyc-I and 6-cyc-I within 5 min of irradiation ( Figure 2). In compound 5, bearing a secondary amine, only a trace of 5-cyc-I was detected overnight. However, the downfield shifted 31 P-NMR signals of cyclic intermediates 4-cyc-I, 5-cyc-I, and 6-cyc-I (δP 28.0, 26.5, 21.7 ppm, respectively) indicated that the amine cargo was still attached to the phosphorus and that phenol was released instead. 31 P-NMR spectra recorded overnight suggested three different scenarios: (1) preferential release of phenol as 4-cyc-I and 4-P2 were detected; (2) formation of the stable intermediate 5-I, which released phenol in several days; (3) sequential release of phenol in minutes (6-cyc-I) and aniline overnight (6-cyc-P). The formation of phospholane intermediates (4-6)-cyc-I could be useful for other applications, such as preparing functional biopolymers for controlled drug delivery systems [23], but linkers 1-6 did not release amine successfully. Therefore, we altered the spacer structure to find an effective system for releasing amine-containing cargos. The self-immolation of 1-6 was triggered photochemically (365 nm), and the reaction was monitored by 31 P-NMR spectroscopy. Surprisingly, the successful photoactivation of 1-3 yielded intermediates (1-3)-I, with no further spectral change over several days, thus indicating that the cargo was not released ( Figure S1 in Supplementary Materials).
In contrast to 1-3, linkers 4 and 6 afforded cyclic intermediates 4-cyc-I and 6-cyc-I within 5 min of irradiation ( Figure 2). In compound 5, bearing a secondary amine, only a trace of 5-cyc-I was detected overnight. However, the downfield shifted 31 P-NMR signals of cyclic intermediates 4-cyc-I, 5-cyc-I, and 6-cyc-I (δ P 28.0, 26.5, 21.7 ppm, respectively) indicated that the amine cargo was still attached to the phosphorus and that phenol was released instead. 31 P-NMR spectra recorded overnight suggested three different scenarios: (1) preferential release of phenol as 4-cyc-I and 4-P2 were detected; (2) formation of the stable intermediate 5-I, which released phenol in several days; (3) sequential release of phenol in minutes (6-cyc-I) and aniline overnight (6-cyc-P). The formation of phospholane intermediates (4-6)-cyc-I could be useful for other applications, such as preparing functional biopolymers for controlled drug delivery systems [23], but linkers 1-6 did not release amine successfully. Therefore, we altered the spacer structure to find an effective system for releasing amine-containing cargos. The self-immolation of 1-6 was triggered photochemically (365 nm), and the reaction was monitored by 31 P-NMR spectroscopy. Surprisingly, the successful photoactivation of 1-3 yielded intermediates (1-3)-I, with no further spectral change over several days, thus indicating that the cargo was not released ( Figure S1 in Supplementary Materials).
In contrast to 1-3, linkers 4 and 6 afforded cyclic intermediates 4-cyc-I and 6-cyc-I within 5 min of irradiation ( Figure 2). In compound 5, bearing a secondary amine, only a trace of 5-cyc-I was detected overnight. However, the downfield shifted 31 P-NMR signals of cyclic intermediates 4-cyc-I, 5-cyc-I, and 6-cyc-I (δP 28.0, 26.5, 21.7 ppm, respectively) indicated that the amine cargo was still attached to the phosphorus and that phenol was released instead. 31 P-NMR spectra recorded overnight suggested three different scenarios: (1) preferential release of phenol as 4-cyc-I and 4-P2 were detected; (2) formation of the stable intermediate 5-I, which released phenol in several days; (3) sequential release of phenol in minutes (6-cyc-I) and aniline overnight (6-cyc-P). The formation of phospholane intermediates (4-6)-cyc-I could be useful for other applications, such as preparing functional biopolymers for controlled drug delivery systems [23], but linkers 1-6 did not release amine successfully. Therefore, we altered the spacer structure to find an effective system for releasing amine-containing cargos.

Lactate Phosphate-Based Linkers
To stimulate amine cargo release, we altered the glycol spacer in 1-3 to an L-lactate spacer, thus preparing linkers 7-9 (Scheme 2). Despite promoting a slow release of phenolic compounds, [25] the lactate spacer could be suitable for amines. Compounds 7-9 were synthesized from DMNB ester 24 via acid-catalyzed esterification in refluxing toluene [25], and intermediate 25 was generated in a reaction of 24 with dichloridate 20 in toluene. The reaction was monitored by 31 P-NMR, and a new pair of 31 P-NMR signals at δ P 4.6 ppm and δ P 4.1 ppm (ca. 1:1 ratio), corresponding to two diastereoisomers of 25, was observed due to the new stereogenic center on phosphorus. Intermediate 25 was directly used for amine phosphorylation, and the final products 7-9 were isolated with good yields (42-52%) as 1:1 mixtures of diastereoisomers, as shown by the two sets of NMR signals.

Lactate Phosphate-Based Linkers
To stimulate amine cargo release, we altered the glycol spacer in 1-3 to an L-lactate spacer, thus preparing linkers 7-9 (Scheme 2). Despite promoting a slow release of phenolic compounds, [25] the lactate spacer could be suitable for amines. Compounds 7-9 were synthesized from DMNB ester 24 via acid-catalyzed esterification in refluxing toluene [25], and intermediate 25 was generated in a reaction of 24 with dichloridate 20 in toluene. The reaction was monitored by 31 P-NMR, and a new pair of 31 P-NMR signals at δP 4.6 ppm and δP 4.1 ppm (ca. 1:1 ratio), corresponding to two diastereoisomers of 25, was observed due to the new stereogenic center on phosphorus. Intermediate 25 was directly used for amine phosphorylation, and the final products 7-9 were isolated with good yields (42-52%) as 1:1 mixtures of diastereoisomers, as shown by the two sets of NMR signals. The SI of 7-9, monitored by 31 P-NMR, showed successful amine release, which provided the final product P in 15 min ( Figure S2 in the Supplementary Materials). However, in 7 and 9, we also detected a new 31 P signal (δP −1.6 ppm) belonging to the undesired product of alternative decomposition (7-X and 9-X). Interestingly, linker 8, bearing a secondary amine as a cargo, did not form the undesired product 8-X and followed the expected reaction course.
Given the unexpected reactivity of 7 and 9 in the CACO/DMSO mixture (1/1, v/v), we optimized the solvent system. For this purpose, we performed irradiation experiments on 7, in various solvent mixtures ( Figure S3 in the Supplementary Materials), and we found that the formation of side product 7-X can be suppressed by either decreasing the pH of the cacodylate buffer (to pH = 5) or changing the buffer itself. HEPES buffer or an unbuffered system can suppress the formation of 7-X. Lastly, we selected the HEPES (pH 7.4)/DMSO system (1:1, v/v) for further SI investigation.
We monitored the SI of 7-9 in the HEPES/DMSO solvent mixture ( Figure 3). In 5 min, we detected the final product P in all cases (δP −1.1 ppm). Linkers 7 and 8 did not provide any intermediate 7-I and 8-I, respectively, as they undergo fast cyclization, and photoactivation is a rate-limiting step. In turn, the SI of 9 was slow, and we did detect intermediate 9-I ( Figure 3, right) or traces of the undesired product 9-X (δP −1.6 ppm). Considering the overall limited stability of 7 and 9, we investigated the formation of the unknown side product X and performed stability tests in 7-9. The results showed the limited stability of 7 and 9, which were significantly decomposed within 7 days (Figures S6 and S7 in the Supplementary Materials, details therein). The SI of 7-9, monitored by 31 P-NMR, showed successful amine release, which provided the final product P in 15 min ( Figure S2 in the Supplementary Materials). However, in 7 and 9, we also detected a new 31 P signal (δ P −1.6 ppm) belonging to the undesired product of alternative decomposition (7-X and 9-X). Interestingly, linker 8, bearing a secondary amine as a cargo, did not form the undesired product 8-X and followed the expected reaction course.
Given the unexpected reactivity of 7 and 9 in the CACO/DMSO mixture (1/1, v/v), we optimized the solvent system. For this purpose, we performed irradiation experiments on 7, in various solvent mixtures ( Figure S3 in the Supplementary Materials), and we found that the formation of side product 7-X can be suppressed by either decreasing the pH of the cacodylate buffer (to pH = 5) or changing the buffer itself. HEPES buffer or an unbuffered system can suppress the formation of 7-X. Lastly, we selected the HEPES (pH 7.4)/DMSO system (1:1, v/v) for further SI investigation.
We monitored the SI of 7-9 in the HEPES/DMSO solvent mixture ( Figure 3). In 5 min, we detected the final product P in all cases (δ P −1.1 ppm). Linkers 7 and 8 did not provide any intermediate 7-I and 8-I, respectively, as they undergo fast cyclization, and photoactivation is a rate-limiting step. In turn, the SI of 9 was slow, and we did detect intermediate 9-I ( Figure 3, right) or traces of the undesired product 9-X (δ P −1.6 ppm). Considering the overall limited stability of 7 and 9, we investigated the formation of the unknown side product X and performed stability tests in 7-9. The results showed the limited stability of 7 and 9, which were significantly decomposed within 7 days (Figures S6 and S7 in the Supplementary Materials, details therein).

Characterization of the Undesired Product X
To identify the alternative decomposition pathway, we characterized the undesired product X. The significant upfield shift (δ P < 0) suggested that the P-NH bond had been cleaved. In addition, one singlet 31 P-NMR signal indicated the lack of a stereogenic center on the phosphorus atom. 31 P-NMR chemical shifts of 7-X and 9-X differed slightly Molecules 2021, 26, 5160 5 of 20 (δ P −1.66 and −1.58 ppm, respectively), as found in the starting compounds 7 and 9 (differing in LGs). Combined, these findings demonstrate that 7-X and 9-X also differ in the amine moiety, which may be explained by the intramolecular rearrangement proposed in Figure 4. Additional 2D NMR experiments performed on linker 7 suggested the formation of carboxamide 7-X. We found the key interaction between the phenethylamine alkyl chain and lactate carbonyl in the HMBC spectrum ( Figure 4). The proposed structure of 7-X was confirmed by HR-MS ( Figure S64 in the Supplementary Materials).

Characterization of the Undesired Product X
To identify the alternative decomposition pathway, we characterized the undesired product X. The significant upfield shift (δP < 0) suggested that the P-NH bond had been cleaved. In addition, one singlet 31 P-NMR signal indicated the lack of a stereogenic center on the phosphorus atom. 31 P-NMR chemical shifts of 7-X and 9-X differed slightly (δP −1.66 and −1.58 ppm, respectively), as found in the starting compounds 7 and 9 (differing in LGs). Combined, these findings demonstrate that 7-X and 9-X also differ in the amine moiety, which may be explained by the intramolecular rearrangement proposed in Figure  4. Additional 2D NMR experiments performed on linker 7 suggested the formation of carboxamide 7-X. We found the key interaction between the phenethylamine alkyl chain and lactate carbonyl in the HMBC spectrum ( Figure 4). The proposed structure of 7-X was confirmed by HR-MS ( Figure S64 in the ESI).
Only the linkers containing the NH group in the phosphoramidate bond underwent the proposed intramolecular rearrangement (linker 8 without NH did not form 8-X). Indeed, N-methylation of 9 yielded linker 10, which did not form the undesired product 10-X ( Figure S4 in the ESI). This intramolecular rearrangement has already been reported by the Mulliez group in 1985 [26].

Characterization of the Undesired Product X
To identify the alternative decomposition pathway, we characterized the undesired product X. The significant upfield shift (δP < 0) suggested that the P-NH bond had been cleaved. In addition, one singlet 31 P-NMR signal indicated the lack of a stereogenic center on the phosphorus atom. 31 P-NMR chemical shifts of 7-X and 9-X differed slightly (δP −1.66 and −1.58 ppm, respectively), as found in the starting compounds 7 and 9 (differing in LGs). Combined, these findings demonstrate that 7-X and 9-X also differ in the amine moiety, which may be explained by the intramolecular rearrangement proposed in Figure  4. Additional 2D NMR experiments performed on linker 7 suggested the formation of carboxamide 7-X. We found the key interaction between the phenethylamine alkyl chain and lactate carbonyl in the HMBC spectrum ( Figure 4). The proposed structure of 7-X was confirmed by HR-MS ( Figure S64 in the ESI).
Only the linkers containing the NH group in the phosphoramidate bond underwent the proposed intramolecular rearrangement (linker 8 without NH did not form 8-X). Indeed, N-methylation of 9 yielded linker 10, which did not form the undesired product 10-X ( Figure S4 in the ESI). This intramolecular rearrangement has already been reported by the Mulliez group in 1985 [26].  Only the linkers containing the NH group in the phosphoramidate bond underwent the proposed intramolecular rearrangement (linker 8 without NH did not form 8-X). Indeed, N-methylation of 9 yielded linker 10, which did not form the undesired product 10-X ( Figure S4 in the Supplementary Materials). This intramolecular rearrangement has already been reported by the Mulliez group in 1985 [26].

Amine Screening-Application Scope
Based on the successful SI observed in linkers 7-9, we examined the synthetic scope and application feasibility of lactate linkers by altering the structure of the cargo. Accordingly, we prepared linkers 10-16 (Scheme 3). final products 11 and 12, respectively. Aromatic amines provided only two linkers bearing N-methylaniline and 2-aminopyrimidine (10 and 13, respectively). Other heterocyclic amines, such as imidazole, indoline, 2-aminobenzothiazole, 1-and 2-aminobenzoimidazole, and 2-aminobenzoxazole, yielded complex reaction mixtures, as shown in 31 P-NMR spectra ( Figure S9 in the Supplementary Materials), which we were unable to separate.
Since cargo release was faster in 4-6, phenyl-lactyl phosphate analogs 14-16 were also prepared with a phenyl instead of an ethyl group attached to the phosphorus. Then, we subjected 10-16 to irradiation NMR experiments (Figures 5 and 6). Compounds 10-12 afforded the final product P within 5 min, and product P was a major component in the reaction mixtures in 15 min. In contrast, 2-aminopyrimidine derivative 13 showed a slow formation of intermediate 13-I without any further spectral change in 15 min. Lastly, the pyrimidine cargo was fully released from 13-I within 19 days.  Linkers with aliphatic amines (morpholine and benzylamine) were synthesized as final products 11 and 12, respectively. Aromatic amines provided only two linkers bearing Nmethylaniline and 2-aminopyrimidine (10 and 13, respectively). Other heterocyclic amines, such as imidazole, indoline, 2-aminobenzothiazole, 1-and 2-aminobenzoimidazole, and 2aminobenzoxazole, yielded complex reaction mixtures, as shown in 31 P-NMR spectra ( Figure S9 in the Supplementary Materials), which we were unable to separate.
Since cargo release was faster in 4-6, phenyl-lactyl phosphate analogs 14-16 were also prepared with a phenyl instead of an ethyl group attached to the phosphorus.
Then, we subjected 10-16 to irradiation NMR experiments (Figures 5 and 6). Compounds 10-12 afforded the final product P within 5 min, and product P was a major component in the reaction mixtures in 15 min. In contrast, 2-aminopyrimidine derivative 13 showed a slow formation of intermediate 13-I without any further spectral change in 15 min. Lastly, the pyrimidine cargo was fully released from 13-I within 19 days.

Amine Screening-Application Scope
Based on the successful SI observed in linkers 7-9, we examined the synthetic scope and application feasibility of lactate linkers by altering the structure of the cargo. Accordingly, we prepared linkers 10-16 (Scheme 3).
Since cargo release was faster in 4-6, phenyl-lactyl phosphate analogs 14-16 were also prepared with a phenyl instead of an ethyl group attached to the phosphorus. Then, we subjected 10-16 to irradiation NMR experiments (Figures 5 and 6). Compounds 10-12 afforded the final product P within 5 min, and product P was a major component in the reaction mixtures in 15 min. In contrast, 2-aminopyrimidine derivative 13 showed a slow formation of intermediate 13-I without any further spectral change in 15 min. Lastly, the pyrimidine cargo was fully released from 13-I within 19 days.  Linkers 14-16 released their amine cargos slightly faster than their ethyl counterparts 7-9. Linkers 14-16 afforded the final product P, which emerged as one singlet 31 P signal at δ P − 6 ppm ( Figure 6). Although 14 and 15 released the corresponding amines within 5 min, linker 16 did so overnight. Linkers 14-16 released their amine cargos slightly faster than their ethyl counterparts 7-9. Linkers 14-16 afforded the final product P, which emerged as one singlet 31 P signal at δP −6 ppm ( Figure 6). Although 14 and 15 released the corresponding amines within 5 min, linker 16 did so overnight.

Application
Ultimately, we prepared two model prodrugs of Ciprofloxacin (Figure 7), which is a known fluoroquinolone antibiotic containing a secondary amino group, to demonstrate the applicability of lactate phosphate-based linkers. To avoid side reactions during the synthesis of 17 and 18, we protected the carboxylic group of Ciprofloxacin by methylation, which should not decrease the antibiotic activity as reported previously [27]. Then, Ciprofloxacin methyl ester 27 was phosphorylated, following the procedure that had been used for model linkers 7-9 (Scheme 2). Two-step phosphorylation starting from DMNB ester 24 and ethyl dichlorophosphate 20 afforded photoactivable compound 17. In addition to 17, we also prepared its enzymatically activable analog 18. Both compounds were obtained in moderate isolated yields (43-44%) as ca. 1:1 mixtures of diastereoisomers, as confirmed by the presence of two sets of NMR signals. The photoactivation of linker 17 resulted in Ciprofloxacin release in 5 min, which was supported by the formation of product 17-P (Figure 8a). Compound 18 was activated by a lipase from Candida Antarctica. We detected 18-P after 3 h, with ca. 30% cargo release in 24 h. Most Ciprofloxacin (97%) was released in 6 days (Figure 8b). Enzymatic cargo release was relatively slow, which was presumably due to the substrate specificity of the lipase that was used in the experiment.
Furthermore, we performed a biological screening of 18 and 27, which showed that Ciprofloxacin methylation at the carboxylic moiety inhibits the antibiotic activity of this

Application
Ultimately, we prepared two model prodrugs of Ciprofloxacin (Figure 7), which is a known fluoroquinolone antibiotic containing a secondary amino group, to demonstrate the applicability of lactate phosphate-based linkers. To avoid side reactions during the synthesis of 17 and 18, we protected the carboxylic group of Ciprofloxacin by methylation, which should not decrease the antibiotic activity as reported previously [27]. Then, Ciprofloxacin methyl ester 27 was phosphorylated, following the procedure that had been used for model linkers 7-9 (Scheme 2). Two-step phosphorylation starting from DMNB ester 24 and ethyl dichlorophosphate 20 afforded photoactivable compound 17. In addition to 17, we also prepared its enzymatically activable analog 18. Both compounds were obtained in moderate isolated yields (43-44%) as ca. 1:1 mixtures of diastereoisomers, as confirmed by the presence of two sets of NMR signals. Linkers 14-16 released their amine cargos slightly faster than their ethyl counterparts 7-9. Linkers 14-16 afforded the final product P, which emerged as one singlet 31 P signal at δP −6 ppm ( Figure 6). Although 14 and 15 released the corresponding amines within 5 min, linker 16 did so overnight.

Application
Ultimately, we prepared two model prodrugs of Ciprofloxacin (Figure 7), which is a known fluoroquinolone antibiotic containing a secondary amino group, to demonstrate the applicability of lactate phosphate-based linkers. To avoid side reactions during the synthesis of 17 and 18, we protected the carboxylic group of Ciprofloxacin by methylation, which should not decrease the antibiotic activity as reported previously [27]. Then, Ciprofloxacin methyl ester 27 was phosphorylated, following the procedure that had been used for model linkers 7-9 (Scheme 2). Two-step phosphorylation starting from DMNB ester 24 and ethyl dichlorophosphate 20 afforded photoactivable compound 17. In addition to 17, we also prepared its enzymatically activable analog 18. Both compounds were obtained in moderate isolated yields (43-44%) as ca. 1:1 mixtures of diastereoisomers, as confirmed by the presence of two sets of NMR signals. The photoactivation of linker 17 resulted in Ciprofloxacin release in 5 min, which was supported by the formation of product 17-P (Figure 8a). Compound 18 was activated by a lipase from Candida Antarctica. We detected 18-P after 3 h, with ca. 30% cargo release in 24 h. Most Ciprofloxacin (97%) was released in 6 days (Figure 8b). Enzymatic cargo release was relatively slow, which was presumably due to the substrate specificity of the lipase that was used in the experiment.
Furthermore, we performed a biological screening of 18 and 27, which showed that Ciprofloxacin methylation at the carboxylic moiety inhibits the antibiotic activity of this The photoactivation of linker 17 resulted in Ciprofloxacin release in 5 min, which was supported by the formation of product 17-P (Figure 8a). Compound 18 was activated by a lipase from Candida Antarctica. We detected 18-P after 3 h, with ca. 30% cargo release in 24 h. Most Ciprofloxacin (97%) was released in 6 days (Figure 8b). Enzymatic cargo release was relatively slow, which was presumably due to the substrate specificity of the lipase that was used in the experiment.
Furthermore, we performed a biological screening of 18 and 27, which showed that Ciprofloxacin methylation at the carboxylic moiety inhibits the antibiotic activity of this fluoroquinolone (Table S1 in the Supplementary Materials), despite previous reports stating otherwise [27]. Nevertheless, we believe that phosphate-based linkers may be used to design secondary amine drug delivery systems.  (Table S1 in the Supplementary Materials), despite previous reports stating otherwise [27]. Nevertheless, we believe that phosphate-based linkers may be used to design secondary amine drug delivery systems.

Discussion
Our study demonstrates that a universal spacer for delivering all types of amines will unlikely ever be designed given the sensitivity of the phosphorus atom to substitution. An ethylene glycol spacer, which was previously identified as the best linker for phenolic cargo delivery [22], proved inefficient in delivering amines, as shown in 1-6. An alteration in the electron density of phosphorus caused by oxygen substitution (from phenol-previous work [22]) for nitrogen (from amine) could explain the inefficiency of 1-6. Although installing phenol (4-6) as the second cargo slightly accelerated the SI, when compared to the ethyl analogs 1-3, phenol was preferentially released instead of the amine cargos. Surprisingly, the L-lactate spacer was the most suitable for aliphatic amines, releasing the cargo in 15 min (7, 8, 11, 12, 14, 15). In contrast, linkers with aromatic amine cargos (aniline (9 and 16), N-methylaniline (10), and 2-aminopyrimidine (13)) released their cargos more slowly than linkers bearing aliphatic amines, as indicated by the formation of higher amounts of intermediate I.
Slower SI, especially in 13 (13-I), could be partly explained by the low pKa of 2-aminopyrimidine (pKa 3.54 [28]). Although there is no clear correlation between the pKa of amine and the SI rate, pKa plays a key role in amine release [29]. N-protonation in phosphoramidates facilitates the nucleophilic attack of water (a carboxylate group in our case) to the phosphorus atom. Imbach [30] has shown that phosphoramidates consisting of low pKa amines are stable, whereas those containing amines with a higher pKa (more than 9) show the fastest hydrolysis, which has also been described by Wagner [31]. Nevertheless, differences in cargo release rate could not be easily explained by the various pKa of amines. Based on Mayr's extensive studies of amine behavior in solution, the amine leaving group can be affected by attributes other than pKa, such as nucleophilicity [32], hydration energy (amine stabilization by solvation in water), polarity, and solvent pH, in addition to structural (cyclic vs. acyclic) or sterical effects. Therefore, predicting the optimal spacer for a specific cargo is a difficult task, and designing purposeful drug delivery systems requires studying structure-activity relationships in detail.

Discussion
Our study demonstrates that a universal spacer for delivering all types of amines will unlikely ever be designed given the sensitivity of the phosphorus atom to substitution. An ethylene glycol spacer, which was previously identified as the best linker for phenolic cargo delivery [22], proved inefficient in delivering amines, as shown in 1-6. An alteration in the electron density of phosphorus caused by oxygen substitution (from phenol-previous work [22]) for nitrogen (from amine) could explain the inefficiency of 1-6. Although installing phenol (4-6) as the second cargo slightly accelerated the SI, when compared to the ethyl analogs 1-3, phenol was preferentially released instead of the amine cargos. Surprisingly, the L-lactate spacer was the most suitable for aliphatic amines, releasing the cargo in 15 min (7, 8, 11, 12, 14, 15). In contrast, linkers with aromatic amine cargos (aniline (9 and 16), N-methylaniline (10), and 2-aminopyrimidine (13)) released their cargos more slowly than linkers bearing aliphatic amines, as indicated by the formation of higher amounts of intermediate I.
Slower SI, especially in 13 (13-I), could be partly explained by the low pK a of 2aminopyrimidine (pK a 3.54 [28]). Although there is no clear correlation between the pK a of amine and the SI rate, pK a plays a key role in amine release [29]. N-protonation in phosphoramidates facilitates the nucleophilic attack of water (a carboxylate group in our case) to the phosphorus atom. Imbach [30] has shown that phosphoramidates consisting of low pK a amines are stable, whereas those containing amines with a higher pK a (more than 9) show the fastest hydrolysis, which has also been described by Wagner [31]. Nevertheless, differences in cargo release rate could not be easily explained by the various pK a of amines. Based on Mayr's extensive studies of amine behavior in solution, the amine leaving group can be affected by attributes other than pK a , such as nucleophilicity [32], hydration energy (amine stabilization by solvation in water), polarity, and solvent pH, in addition to structural (cyclic vs. acyclic) or sterical effects. Therefore, predicting the optimal spacer for a specific cargo is a difficult task, and designing purposeful drug delivery systems requires studying structure-activity relationships in detail.

Materials and Methods
Unless otherwise indicated, all chemicals were purchased from commercial suppliers (Sigma Aldrich, Merck, EU; Fluorochem, UK; Acros Organics, Thermo Fisher Scientific, EU) and used without further purification. All reactions sensitive to air or moisture were performed under an inert atmosphere of argon in dry solvents. Thin layer chromatography (TLC) was performed on TLC aluminium sheets (silica-gel 60 F 254 ; Merck, EU) and visualized by UV fluorescence. The reaction was monitored by TLC and/or 31 P-NMR spectroscopy in CDCl 3 . Flash-column chromatography was performed on a Compact (ECOM s.r.o., EU) chromatography system using silica-gel or C18 silica-gel 230-400 mesh, 60 Å (Merck KGaA, EU).
NMR spectroscopy. NMR spectra were recorded on a Bruker Avance III spectrometer operating at 400 MHz for 1 H and 101 MHz for 13 C equipped with a probe with an ATM module (5 mm BBFO BB-19F/1H/D Z-GRD). For NMR signal assignment, standard Bruker pulse sequences were used for 1D ( 1 H, 13 C-APT, 31 P, 19 F) and 2D (COSY, ROESY, HSQC, HMBC) NMR experiments at a corrected temperature of 25 • C. NMR spectra coupled with UV irradiation were recorded on a Bruker Avance III spectrometer with a broad-band cryo probe with an ATM module (5 mm CPBBO BB-1H/19F/15N/D Z-GRD) operating at 500 MHz for 1 H and 125.7 MHz for 13 C. All NMR data were interpreted using Topspin 3.5. For reference, the following solvent signals were used: DMSO-d 6 : 2.50 ( 1 H) and 39.7 ( 13 C) ppm or CDCl 3 : 7.28 ( 1 H) and 77.0 ( 13 C) ppm. The 31 P-NMR spectra were referenced to H 3 PO 4 with 0 ppm.
For NMR experiments with in situ irradiation, a light emitting diode (LED; Thorlabs, EU) was used at 365 nm. The light was guided into the spectrometer, directly into the NMR tube via a multimode silica optical fiber with 1 mm diameter, 0.39 NA, and a high amount of OH (Thorlabs, EU).
All products were viscous oils, semi-solids or non-crystalline solids. The reaction conditions were not optimized for the highest possible yields.
General Procedure for the One-Pot Synthesis of Phosphate-Based Linkers. For each experiment, a DMNB-containing photoarm 19 or 24 (0.5 mmol, 1.0 eq.) was dissolved in 2.5 mL of dry toluene under argon at 25 • C, adding dry TEA (90.6 µL, 0.65 mmol, 1.3 eq.) followed by the corresponding dichlorophosphate (0.5 mmol, 1.0 eq.). The reaction mixture was stirred at 25 • C for 16 h, and the formation of the intermediates was confirmed by 31 P-NMR (22: δ P 4.7 ppm; 23: δ P 0.08 ppm; 25: δ P 4.6 and 4.1 ppm; 26: δ P −0.08 and −0.17 ppm) before phosphorylating the amines. The corresponding amine (0.5 mmol, 1.0 eq.) was added, followed by dry TEA (69.7 µL, 0.5 mmol, 1.0 eq.). The reaction mixture was stirred at room temperature until completing the reaction (monitored by 31 P-NMR). After evaporating the solvent, pure products were isolated by Flash silica gel chromatography, which was followed by reverse-phase chromatography, as described for each compound.

Conclusions
In summary, we designed and synthesized phosphate-based SI linkers for aminecontaining drug delivery. We found that the lactate spacer can release amines effectively within 15 min; moreover, it can release two cargos sequentially-the first amine cargo within minutes and the second phenolic cargo overnight. Surprisingly, this is exactly the opposite release order that we found when using an ethylene glycol SI spacer, whereby phenol is released preferentially [22]. Interestingly, the linkers bearing primary amines lack stability at physiological pH (pH = 7.4) due to an intramolecular rearrangement caused by the nucleophilic attack of NH nitrogen from LG on the carbonyl group of lactate. This alternative decomposition, which yields the undesired product X, can be suppressed by changing the buffer (e.g., HEPES instead of Cacodylate buffer, pH = 7.4), by decreasing the buffer pH to mildly acidic (pH = 5), or by N-methylation of phosphoramidate nitrogen. In turn, derivatives bearing secondary amines are stable in a range of pH 5-7.4. As such, our prodrug approach is the most suitable for the delivery of secondary amines. Further applicability was demonstrated by phosphorylation of the antibiotic Ciprofloxacin, whose phototriggerable and enzyme-triggerable prodrugs released Ciprofloxacin successfully. Overall, our results establish an experimental paradigm for the smart design of new self-immolative systems for the targeted delivery of various amine-containing drugs and their enhanced cellular uptake and activity, thus broadening the applications of prodrug technology. Moreover, phospholane amidates could lead to the design of new synthetic approaches in phosphorus chemistry.